Each year 15% of couples seek medical advice because of difficulties becoming pregnant (WHO 1997). Sub-fertility is therefore currently one of the most frequent health concerns facing the population aged 25-45 years. For the past two decades, in vitro fertilisation (IVF) has provided an effective form of assistance for a large proportion of these couples. Indeed, IVF now accounts for 1.3% of all live births in Europe (Nygren et al. 2001) and 1.7% of all live births in Australasia (Hurst et al. 2001).
From the inception of routine IVF in 1978, pregnancy rates have risen steadily to levels considered normal for the fertile population (approximately 25% per attempt). The quest to break through this physiological barrier is driven by the significant financial and emotional cost for each IVF treatment for individuals.
Failure of embryos to implant into the lining of the uterus (endometrium) during an IVF treatment cycle is widely accepted by health professionals as the most significant limiting factor to improving success rates. The scale of embryo wastage following transfer into IVF patients is enormous, such that 80-85% of embryos fail to result in a pregnancy (Blake et al 2002). Recent analysis of daily urine levels of human chronic gonadotrophin (hCG) in women undergoing an IVF cycle, demonstrated that implantation was detected in as many as 60% of the cycles (Simon et a/1999). Of all embryos transferred in an IVF, 40% fail to implant.
There are two broad reasons for failure of implantation following replacement of apparently viable embryos. The first involves intrinsic embryonic factors that reflect retarded development or deficiencies in the health of the blastocyst itself and its ability to hatch (Gott et al 1990, Plachot 1992, van Kooij et al 1996). The second relates to extrinsic factors that imply a lack of implantation receptivity in the endometrium (Edwards 1986, Yaron 1994). Moreover, successful implantation is dependent on the synchrony of embryonic development and endometrial maturation that is largely controlled by the ovarian hormone milieu.
Recently it has become apparent that fertility drugs used for the super-ovulation of women undergoing IVF are predominantly responsible for the compromised implantation receptivity observed on both sides of the embryonic/maternal interface. Ertzeid and Storeng demonstrated the detrimental effects of gonadotropins on implantation using a series of cross-over embryo transfer experiments (Ertzeid et al. 2001). Embryos from super-ovulated and non-stimulated females were transferred to separate uterine horns in the same super-ovulated or non-stimulated pseudo-pregnant recipient mice. A significant decrease in implantation was observed in the uterine horns receiving embryos from super-ovulated donors and even more dramatically in both horns of super-ovulated recipients.
Highly elevated concentrations of estrogen result from ovarian stimulation in IVF. These are suspected to alter the cascade of hormonal events and subsequent expression of cytokines that the oocytes, embryos and uterine endometrium would ordinarily be exposed to in an unstimulated menstrual cycle. Add to this the physiological challenge of in vitro culture, largely devoid of growth factors, and it is not unexpected that IVF derived embryos might be compromised at the time of implantation.
Despite substantial advances in the recovery and maturation of multiple occytes from unstimulated cycles, the practice of oocyte in vitro maturation (IVM) is as yet clinically unaccepted. With the prospect that super-ovulation will remain the mainstay of IVF, other approaches to improving implantation rates continue to be explored.
The development of physiological based culture media constituents has gone some way to improving the development of embryos in culture for up to 6 days. This extended culture enables self-selection of the most viable embryos for transfer, but as a consequence this approach has a high attrition rate of embryos. Co-culture of embryos on a mono- or bi-layer of support cells (e.g. endometrial cells) has also provided a method for improving the development of embryos in culture presumably via the stimulus of growth factors. More directly the addition of a variety of growth factors to media has been explored and shown to be of benefit (Sjoblom et al. 2000).
Maintaining a receptive endometrium through administration of human chorionic gonadotropin or progesterone has been practiced since the early days of IVF. In fact only after additional progesterone support was given in the luteal phase of the cycle, did the world's first IVF pregnancies result. It has long been recognised that the elevated estrogen profiles produced by the fertility drugs effectively advance the endometrial tissue dating by approximately one day (Noyes et al., 1950; Pittaway et al. 1983; Garcia et al. 1984). Compound this with the fact that embryos are routinely transferred into the uterus at the 2-8 cell stage (48-72 hrs prematurely to what occurs naturally) and it is clear that IVF results in an asynchronous environment for implantation.
Implantation of a hatched blastocyst is described as consisting of three phases:                a) apposition— where the embryo comes into initial physical contact with the glycoconjugate coat of the endometrial epithelium (called the glycocalyx).        b) adhesion— where the embryo undergoes cell to cell, and cell to matrix binding with molecules derived from the apical cells on the endometrium.        c) invasion— where the embryo penetrates through the epithelial layer of the endometrium by intruding between cell junctions as occurs in the human or by displacement of the cells found in some animals (e.g. mice).        
Super-ovulation has been postulated to alter electronegative properties of the glycocalyx and apical cell surface of the endometrium. In this way; fertility drugs may reduce effective apposition and adhesion of a transferred embryo (Ronnberg et al. 1985).
At least two therapeutic approaches to improving implantation rates in IVF embryos have been practiced in humans. The first draws on the observation that inclusion of the glycoaminoglycan, hyaluronan, in the media containing embryos for transfer, results in a higher implantation rate than media devoid of this polysaccharide (Gardner et al. 1999). The concentration of hyaluronan increases in the uterus at the time of implantation in the mouse (Zorn et al. 1995) and is suggested to facilitate implantation by a variety of means such as increasing cell-cell and cell-matrix adhesion and indirectly through promotion of angiogenesis. Despite a lack of published trials in humans, hyaluronate is now present in a number of commercially available embryo transfer media.
One therapy that has undergone clinical trials and is described in U.S. Pat. No. 6,196,965, is the use of a fibrin sealant. The first experiments with a fibrin sealant were carried out in 1981, and by 1988 it had been proven safe to use in humans (Rodrigues et al. 1988).
U.S. Pat. No. 6,196,965 is based on the technique used in a randomised clinical trial published in 1992 (Feichtinger et al. 1992). Embryos are transferred in a catheter, sandwiched between small quantities of thrombin/aprotinin and then fibrin. The results of the trial demonstrated no significant difference in pregnancy rate between the control and treatment group (546 patients), but a significant decrease in ectopic pregnancies in the fibrin sealant group.
The rationale and theoretical basis for the two therapeutic approaches described above are different. Hyaluronate is added to transfer media in the hope that it will induce a more physiologically receptive environment for implantation. There is, however, an absence of direct evidence at the molecular level proving this hypothesis. Fibrin sealant therapy on the other hand, is used to encase the embryos in an adhesive plug that will theoretically be glued onto the endometrium. Expulsion of embryos from the uterine cavity by muscular contraction and avoidance of ectopic pregnancy was the predominant motivation for the fibrin sealant in the Feichtinger trial (Feichtinger et al. 1990), although other investigators have hypothesised that fibrin would improve the adhesion phase of implanting embryos (Rodrigues et al. 1988).
In addition to the previously described therapeutic approaches, the specification for international application no. PCT/US98/15124 (published as WO 99/05255) describes the enhancement of implantation by contacting the embryo with a lipid-modified adhesion molecule so as to modify the development of the embryo. The technique of “protein painting” embryos with glycosylphosphatidylinositol (GPI) linked Qa-2 proteins to increase the cell division rate is described.
Protein painting is a method for modifying the external antigens of cell membranes without gene transfer. The method exploits the ability of GPI linked proteins to spontaneously anchor to cell membrane via their lipid tails. The method described in the specification for international application no. PCT/US98/15124 (WO 99/05255) requires that a naturally occurring (or genetically altered) protein is inserted into an embryo membrane with an attached GPI lipid tail. Isolated GPI-anchored proteins are stated as having an unusual capacity to reintegrate with a cell-surface membrane. The molecules that can be used for modifying an embryo in this way are therefore confined to a rather limited group.
As described herein, the inventors have now found that embryos can be modified with a range of selected synthesised molecules (modified glycolipids and glycolipid-attachment molecule constructs) and have the ability to bind with mucus, and/or mucus components, and/or cell membranes. The molecules are prepared exogenously by chemical or biological processes.
Not only has the modification of embryos by the method of the invention been successfully demonstrated in an in vitro culture system, but animals have given birth to healthy offspring derived from modified embryos. Embryos prepared in accordance with the invention appear to be developmentally indistinguishable from their unmodified counterparts.
It is an object of this invention to provide a modified embryo for the enhanced implantation of the embryo into the endometrium of an animal, or to at least provide the public with a useful choice.